Identification of Binding Proteins of the Xenopus Morphogenetic Factor Tumorhead Tumorhead (TH) is a morphogenetic factor from Xenopus laevis that has been implicated in the regulation of the processes of cell proliferation and differentiation during development. Loss of TH function studies have shown that TH is needed for ectodermal cells to proliferate and later differentiate into neural and epidermal cells. TH overexpression in frogs leads to increased proliferation of ectodermal derivatives and formation of tumor-like structures, while its expression in transgenic mice produces tumors, many of which are ectodermal in origin. In Xenopus embryos, the TH protein shows a dynamic pattern of sub-cellular localization, shuttling from the cell periphery into the nucleus as development proceeds. Blocking TH's nuclear translocation causes embryonic lethality at the early gastrula transition. Recent studies suggest that TH sub-cellular localization is regulated by Xenopus p21-activated kinase 1 (X-PAK1), whose overexpression is also associated with tumor growth. Thus, TH is a positive regulator of cell proliferation, is functionally conserved, and it has to be properly regulated for normal development. However, the molecular mechanism(s) of action of TH is unknown. We propose to identify TH interacting proteins using a mass spectrometry-based proteomics approach, which can lead us to understand how TH functions during early development. We will immunopurify TH from Xenopus embryos. Proteins that co-purify with TH from embryo cell extracts will be visualized in polyacrylamide gels, and will be identified by mass spectrometry. The interaction between TH and the candidate proteins identified using this approach will be validated using traditional coimmunoprecipitation experiments with embryo cell extracts. Binding proteins whose interaction with TH has been validated will be analyzed by several cellular and molecular approaches. We will start the characterization of the candidate proteins by analyzing if their overexpression and depletion causes the same abnormalities in cell proliferation and differentiation that are caused by TH overexpression and depletion. Next, we will do a series of experiments to determine if there is a functional link between the TH interacting proteins and TH. We will first determine if these proteins co-localize with TH during embryogenesis by doing sub-cellular co-localization studies. We will then address if overexpression or depletion of the candidate proteins rescues the normal phenotype after TH has been depleted. Likewise, we will study whether alterations in the levels of the candidate proteins rescue the normal phenotype impaired by TH overexpression. These experiments will lead us to establish if there is a connection between TH and the candidate proteins, and if these proteins are involved in TH's mechanism of action. Our work will be fundamental in obtaining a better understanding of how the processes of cell proliferation and differentiation are regulated during early vertebrate development. PUBLIC HEALTH RELEVANCE: Often, alterations in proteins that regulate cell proliferation and differentiation during embryonic development result in cancer progression later in development. Thus, a proper understanding of how these proteins regulate the processes of cell proliferation and differentiation during embryogenesis is crucial to understand their roles in oncogenesis. An analysis of the regulation of neural cell proliferation and differentiation by the Tumorhead protein in frogs may lead to a better understanding of human neural tumors and the development of potential therapeutic agents.